High-Cell Density Fed-Batch Fermentation Process for Producing Recombinant Protein

ABSTRACT

Methods for producing proteins, for example, recombinant meningococcal 2086 proteins, using fed-batch fermentation with continuous input of an inducer after achieving a threshold parameter, and optionally continuous input of a carbon source, for example, a constant rate input, to improve protein yields, as well as high density protein compositions and compositions for use in the methods of the present invention, are provided.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/833,479, entitled HIGH-CELL DENSITY FED-BATCH FERMENTATIONPROCESS FOR PRODUCING RECOMBINANT PROTEIN, filed on Jul. 27, 2006, thedisclosure of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This application relates generally to novel fed-batch fermentationmethods that provide improved protein expression in bacterial systems,as well as high density protein compositions and compositions that areused in the novel fed-batch fermentation methods.

BACKGROUND OF THE INVENTION

Various fermentation strategies have been used to produce proteins insufficient quantities for laboratory, clinical or commercial use.Fed-batch fermentation has been used to provide increased proteinsyields over those provided by simple batch fermentation methods.Fed-batch fermentation is a process in which, after an initial batchphase, a phase takes place in which one or more nutrients are suppliedto the culture by feeding.

Generally, during the batch phase, cells are initially grown to adesired concentration. At this phase, cell growth is amplified andgenerally no target protein will be produced unless one adds an inducer,such as arabinose, lactose or isopropyl beta-D-thiogalactoside (IPTG),depending on the promoter, or there is some leakage of the promoter.During the feed phase, carbon source and other requirements aretypically fed to a fermentor in a relatively concentrated liquid streamat a certain feed rate. Once a target cell density is achieved, a feedis commenced with the inducer or the inducer and other nutrients. Inthis phase, the emphasis is on protein production by the grown cells.Substrate (that is, the nutrients and the inducer) that is fed to thefermentor is at this stage used generally for cell growth and productsynthesis. The cell growth is controlled by the feed rate to obtain anoptimum cell growth and production of protein. During the proteinproduction stage, an inducer must be added for recombinant organisms.

Protein expression on a medium comprising a common carbon source such asglucose or another sugar based carbon source and an inducer issatisfactory until limiting conditions arise at the end of the feedphase. Examples of limiting conditions include reduced oxygenconcentration, reduced nutrients such as vitamins, carbon, nitrogen andaccumulation of toxic compounds in the growth medium.

Fed-batch fermentation strategies often involve different forms offeedback control, including indirect and direct feedback to control thesupply of nutrients. One such fed-batch fermentation method involvesapplication of a feedback control algorithm by feeding nutrients inorder to control a process parameter at a defined set point. Forexample, direct control of feed may be based on measurement of nutrientconcentration. Feedback control is then directly related to cellactivity throughout fermentation. Control parameters which have beenused for feedback control of fermentations include pH value, on linemeasured cell density or dissolved oxygen tension (DOT).

However, the application of feedback algorithms is accompanied by anumber of disadvantages. One such disadvantage is that the feed ratedepends on current process parameters. Any disturbance to the processmay affect the parameter thus distorting the feed rate and resultingprotein yield. Such disadvantages are magnified as the process isscaled-up to produce increased protein quantities.

Another disadvantage of previously employed fed-batch strategies is thatwhen using feed-back control, the specific growth rate cannot be exactlypredefined or controlled, resulting in suboptimal yields in processes,where the product formation is dependent on growth.

Further, when carbon flux (for example, high glucose concentration) intothe central metabolic pathway exceeds the maximum capacity of theTricarboxylic Acid (TCA) cycle, by-products may accumulate. Theaccumulation of by-products could inhibit cell growth and proteinproduction during fermentation.

Additionally, the various deficiencies of fed-batch fermentation methodsoften result in inefficient use of nutrient components. As such, themethods may be economically disadvantageous, particularly for largescale commercial protein production.

Previous approaches to recombinant protein expression through fed-batchfermentation, as described above, have various deficiencies. Given theimportance of cost-effectively producing sufficient quantities ofprotein for various purposes, there is a need for an efficient fed-batchfermentation method that results in higher cell growth, increasedproduct formation (that is, higher protein yield), and decreasedby-product accumulation.

SUMMARY OF THE INVENTION

The present invention relates to novel fed-batch fermentation methodsfor producing unexpectedly high yields of recombinant protein.

An embodiment of the present invention provides a method for producing arecombinant protein comprising: culturing a recombinant bacterial cellto express a recombinant protein comprising continuously adding a carbonsource to a culture comprising the recombinant bacterial cell andcontinuously adding an inducer to the culture after the culture achievesa threshold parameter; and isolating the recombinant protein from thecell culture.

A further embodiment of the present invention provides a method forproducing a recombinant protein comprising: (a) introducing into abacterial host cell an expression vector encoding a recombinant proteinunder the control of an inducible promoter to form a recombinantbacterial cell; (b) introducing the recombinant bacterial cell into aculture medium to form a cell culture; (c) adding a carbon source to thecell culture as a continuous feed; (d) monitoring cell growth in thecell culture for achievement of a threshold optical density (OD₆₀₀); (e)adding an inducer of the inducible promoter to the cell culture as acontinuous feed once the threshold optical density (OD₆₀₀) is achieved;and (f) harvesting the recombinant protein from the cell culture.

A still further embodiment of the present invention provides a methodfor producing a recombinant protein comprising: culturing a recombinantbacterial cell to express a recombinant protein by continuously addingan inducer to a culture comprising the bacterial cell after the cultureachieves a threshold parameter, wherein the bacterial cell comprises anucleic acid sequence corresponding to a gene of N. meningitidisserogroup B.

According to an even further embodiment, the present invention providesa method for producing a recombinant 2086 protein (rP2086) comprising:(a) introducing into a bacterial host cell an expression vector encodinga recombinant meningococcal 2086 protein under the control of aninducible promoter to form a recombinant bacterial cell; (b) introducingthe recombinant bacterial cell to a culture medium to form a culture;(c) adding a carbon source to the culture; (d) monitoring cell growth inthe culture for achievement of a threshold optical density (OD); (e)continuously adding an inducer of the inducible promoter to the cultureonce the cell density of the culture achieves an optical density ofabout 70 to 110; and (f) harvesting the recombinant meningococcal 2086protein from the culture after about 3 hours to about 6 hours aftercommencement of continuously adding the inducer.

According to another embodiment, the present invention provides acomposition comprising: a bacterial culture comprising a recombinant2086 protein (rP2086) at a density of at least about 1.5 g/L based onthe total volume of the bacterial culture.

According to yet another embodiment, the present invention provides acomposition comprising: a bacterial culture medium comprising arecombinant meningococcal 2086 protein (rP2086) prepared according tothe methods of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Fed-batch fermentation at various constant feed rates withoutinduction.

FIG. 2: Fed-batch fermentation at various constant feed rates withoutinduction.

FIG. 3: Induction at various optical densities.

FIG. 4: Induction with various arabinose levels.

FIG. 5: Effects of arabinose addition method on rLP2086 yield.

FIG. 6: Effect of arabinose feed rate on rLP2086 production.

FIG. 7: Effect of induction time on expression.

FIG. 8: Fed-batch fermentation for subfamily B rLP2086 production.

FIGS. 9 a and 9 b: SDS-PAGE and Western Blot of rLP2086 subfamily Binduction, respectively.

FIG. 10: Fed-batch fermentation for subfamily A rLP2086 production.

FIGS. 11 a and 11 b: SDS-PAGE and Western Blot of rLP2086 subfamily Ainduction, respectively.

FIG. 12 a, 12 b and 12 c: Dual feed of glucose and arabinose duringinduction.

FIG. 13 a: rLP2086 subfamily B E. coli fed-batch fermentation at 100 Lscale.

FIG. 13 b: rLP2086 subfamily A E. coli fed-batch fermentation at 100 Lscale.

FIG. 14 a: rIP2086 subfamily B E. coli fed-batch fermentation with dualglucose and arabinose feed at 100 L scale.

FIG. 14 b: rIP2086 subfamily A E. coli fed-batch fermentation with dualglucose and arabinose feed at 100 L scale.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention are based on the surprisingdiscovery that unexpectedly high protein yields are obtained byfed-batch fermentation with continuous feeding of an inducer duringinduction in a culture medium. Optionally, a carbon source iscontinuously fed prior to and/or during the continuous inducer feed.When induced by arabinose, about 2-3 g/L of a recombinant 2086lipoprotein (rLP2086) (which is expressed by a microorganism having asequence corresponding to the 2086 gene in N. meningitidis serogroup B)was produced in accordance with an embodiment of the invention. Thisrepresents approximately a 2-3-fold increase in rLP2086 yield byfed-batch fermentation for both subfamilies A and B of the 2086 proteincompared to a comparative batch fermentation process. Moreover, themethods of the present invention are readily adaptable to commercialscale production of these and other proteins.

For the purposes of promoting an understanding of the embodimentsdescribed herein, reference will be made to various embodiments andspecific language will be used to describe the same. The terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to limit the scope of the present invention.As used throughout this disclosure, the singular forms “a,” “an,” and“the” include plural reference unless the context clearly dictatesotherwise. Likewise, the singular forms of terms such as “medium”include reference to the plural “media”, and vice versa. Thus, forexample, a reference to “a culture medium” includes a plurality of suchmedia, as well as a single medium; and a reference to “culture medium”includes a plurality of media, as well as a single medium.

The term “inducer”, as used herein, refers to any agent that induces,enhances, or promotes expression of a recombinant protein, whereby geneexpression under the control of the inducible promoter can be directlyregulated by the concentration of that agent.

The term “carbon source”, as used herein, refers to a source of carbonand energy for cells.

The terms “feed”, “fed”, “feeding” or “continuously adding”, as usedinterchangeably herein, refer to adding a substance continuously over aperiod of time rather than all at once. The terms contemplate a singleinitiation and/or termination or multiple start and/or stop points forcontinuously adding the substance during a fermentation process.

The term “recombinant protein”, as used herein, refers to any protein orbiologically active portion thereof (for example, a portion that retainsbiological activity of the full protein) that is not a reporter ormarker gene (for example, a green fluorescent protein) expressed fromrecombinant genetic material encoding amino acids, including peptides,polypeptides, proteins, oligoproteins and/or fusion proteins. Arecombinant protein product may include a therapeutic, prophylactic ordiagnostic product.

METHODS OF THE PRESENT INVENTION

The methods of the present invention provide unexpectedly high yields ofprotein through a novel fed-batch fermentation process involvingcontinuously adding an inducer, such as arabinose, to a culture mediumafter the culture achieves a threshold parameter. A carbon source, suchas glucose, is generally added to a culture comprising a recombinantbacterial cell prior to the induction phase. The carbon source may befed together with the inducer. The inducer may also serve as a secondarycarbon source.

A carbon source, such as glucose, is continuously added to the culturemedium, before and/or during the continuous feed of the inducer to theculture medium, in accordance with an embodiment of the presentinvention. Thus, the continuous feed of the carbon source overlaps thecontinuous feed of the inducer, according to an embodiment. Thecontinuous feed of the carbon source may continue during the entireduration of the continuous inducer feed or only during part(s) of thatduration. In another embodiment, the continuous feed of the carbonsource does not overlap the continuous feed of the inducer. According toan embodiment of the present invention, the inducer and/or carbon sourcemay be fed to the culture at a constant rate.

The fed-batch fermentation process involves several steps resulting inthe production of the desired protein in accordance with an embodimentof the invention. In an initial step, an expression vector that encodesa recombinant protein product under the control of an inducible promoteris prepared and then is introduced into a bacterial host cell. Thebacterial host cell is introduced into a culture medium. An inducer ofthe inducible promoter is fed to the culture (that is, the inducer isadded into the culture continuously over a period of time). The inducermay be fed to the culture at a constant rate. Then, the recombinantprotein product is harvested from the culture. The recombinant proteinproduced in this manner may then be purified as desired and/or used inany suitable manner, such as in a prophylactic, therapeutic ordiagnostic formulation.

High cell density and enhanced protein yield was unexpectedly achievedby fed-batch fermentation with the constant rate feeding of an inducer,which provides a yield of recombinant protein product of approximately a2-3-fold increase as compared to batch fermentation, as illustrated inthe examples provided below. The methods of the present invention areapplicable to large-scale fermentation as well as small-scalefermentation. “Large-scale” fermentation, as used herein, refers tofermentation in a fermentor that is at least approximately 1,000 L involumetric capacity, that is, working volume, leaving adequate room forheadspace. “Small-scale” fermentation refers generally to fermentationin a fermentor that is generally no more than approximately 100 L involumetric capacity, such as 5 L, 10 L, 50 L or 100 L. A demonstratedadvantage of the present fed-batch fermentation process is that it maybe utilized for the production of a recombinant protein product at the5-10 L fermentor scale and is scalable to any volume, for example, 100L, 150 L, 250 L, 500 L, 1000 L or more, without limitation.

Inducers:

The methods described herein relate to the production of recombinantprotein wherein the expression of recombinant protein is under thetranscriptional control of an inducible promoter, whereby geneexpression under the control of the inducible promoter can be directlyregulated by the concentration of the inducer present in the culturemedium. The inducer is continuously provided to a culture medium,optionally at a constant rate. The inducer is added to the culturemedium once a threshold parameter has been achieved. For example, arecombinant protein may be under the control of the araB promoter (forexample, ParaB) that can be directly regulated by the concentration ofarabinose that is added at a constant rate to the culture medium.Suitable inducers for use in conjunction with the present invention arewell known to persons skilled in the art. Examples of inducers of thepresent invention are provided below, without limitation.

Promoter Inducer Arabinose promoter, such as, ParaB Arabinose humanPlasminogen Tumor Necrosis Factor, Activator Inhibitor type-1, Hpai-1TNF Cytochrome P-450 Toxins CYP1A1 Metal-Responsive Element, MRE HeavyMetals, Mouse Mammary Tumor Virus Glucocorticoids Collagenase PhorbolEster Stromolysin Phorbol Ester SV40 Phorbol Ester Proliferin PhorbolEster α-2-Macroglobulin IL-6 Murine MX Gene Interferon, NewcastleDisease Virus Vimectin Serum Thyroid Stimulating Thyroid Hormone Hormoneα. Gene HSP70 Ela, SV40 Large T Antigen Tumor Necrosis Factor FMAInterferon Viral Infection, dsRNA Somatostatin Cyclic AMP FibronectinCyclic AMP lac promoter/operator IPTG

Carbon Source

Any suitable carbon source, for example, glycerol, succinate, lactate,or sugar-based carbon source, for example, glucose, lactose, sucrose,and fructose, is contemplated for use in the present invention, as wouldbe understood by a person of ordinary skill in the art. For example,sugar-based carbon sources that may be used in the present inventioninclude, without limitation, branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturonic acid, D-mannuronic acid (for example, polymannuronicacid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose andneuraminic acid including homopolysaccharides and heteropolysaccharidesfor example, lactose, amylopectin, starch, hydroxyethyl starch, amylose,dextran sulfate, dextran, dextrins, glycogen, or the polysaccharidesubunit of acid mucopolysaccharides, for example, hyaluronic acid;polymers of sugar alcohols such as polysorbitol and polymannitol;heparin or heparan; or any combination thereof, without limitation.Glucose is the primary carbon source according to an embodiment of theinvention. Arabinose, when used as the inducer, may also serve as asecondary carbon source, although it may be the primary carbon source aswell. According to an embodiment, the carbon sources include any ofD-glucose, L-arabinose, sucrose, I-inositol, D-mannitol, β-D-fructose,α-L rhammnose, D-xylose, cellulose, or any combination thereof. One ormore than one carbon source may be used in the present invention.

Bacterial Expression Systems and Plasmids

This invention also provides recombinant bacterial cells comprising anexpression vector, such as a plasmid, comprising an expression controlsequence having promoter sequences and initiator sequences and anucleotide sequence which codes for a desired polypeptide, thenucleotide sequence being located 3′ to the promoter and initiatorsequences. Any suitable expression control sequence and hostcell/cloning vehicle is contemplated, as would be known to a person ofskill in the art based upon the disclosure provided herein.

Suitable expression control sequences and host cell/cloning vehiclecombinations are well known in the art, and are described by way ofexample, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. In general, recombinant DNA techniquesinvolve obtaining by synthesis or isolation a DNA sequence that encodesthe recombinant protein of interest, and introducing it into anappropriate vector/host cell expression system where it is expressed,preferably under the control of an arabinose inducible promoter. Any ofthe methods described for the insertion of DNA into an expression vectormay be used to ligate a promoter and other regulatory control elementsinto specific sites within the selected recombinant vector. Suitablehost cells are then transformed, infected, transduced or transfectedwith such vectors or plasmids by conventional techniques.

A variety of host cell-vector (plasmid) systems may be used to expressthe recombinant protein of interest. The vector system, such as forexample a system including the arabinose inducible promoter, iscompatible with the host cell used. The DNA encoding the recombinantprotein product of interest is inserted into an expression system, andthe promoter (preferably the arabinose inducible promoter), and othercontrol elements are ligated into specific sites within the vector sothat when the vector is inserted into a host cell (by transformation,transduction or transfection, depending on the host cell-vector systemused) the DNA encoding the recombinant protein product of interest isexpressed by the host cell.

The vector may be selected from one of the viral vectors or non-viralvectors described above but must be compatible with the host cell used.The recombinant DNA vector may be introduced into appropriate host cells(bacteria, virus, yeast, mammalian cells or the like) by transformation,transduction or transfection, etc. (depending upon the vector/host cellsystem). Host-vector systems include but are not limited to bacteriatransformed with bacteriophage DNA, plasmid DNA or cosmid DNA.

The expression in prokaryotes of the recombinant protein product ofinterest may be carried out in any suitable species or strain ofbacteria, such as E. coli, with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins.

Fusion vectors add a number of amino acids to a protein encoded therein,to the amino or carboxy terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include Pgex (Pharmacia Biotech Inc;Smith and Johnson, 1988), Pmal (New England Biolabs, Beverly; Mass.) andPrit5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Tightly regulated tac promoter vectorsuseful for the expression of unfused and fused proteins in Escherichiacoli, Gene, 69, 301-315), and Pet lid (Studier et al. (1990) Use of T7RNA polymerase to direct expression of cloned genes, Methods inEnzymology, 185, 60-89). Target gene expression from the pTrc vectorrelies on host RNA polymerase transcription from a hybrid trp-lac fusionpromoter. Target gene expression from the Pet lid vector relies ontranscription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase J7 gn1. This viral polymerase issupplied by host strains BL21 (DE3) or HMS I 74(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

The regulatory sequence of the vector construct is an inducible promoteraccording to an embodiment. The use of an inducible promoter will permitlow basal levels of activated protein to be produced by the cell duringroutine culturing and expansion add. Subsequently, the cells may then beinduced to express large amounts of the desired protein duringproduction or screening. The inducible promoter may be isolated fromcellular or viral genomes.

Inducible promoters that are regulated by exogenously suppliedcompounds, include, without limitation, the arabinose promoter, thezinc-inducible sheep metallothionine (MT) promoter, the dexamethasone(Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7polymerase promoter system (WO 98/10088); the ecdysone insect promoter(No et al., 1996 Proc. Natl. Acad. Sci. USA, 93:3346-3351), thetetracycline-repressible system (Gossen et al., 1992 Proc. Natl. Acad.Sci. USA, 89:5547-5551), the tetracycline-inducible system (Gossen etal., 1995 Science, 268:1766-1769, see also Harvey et al., 1998 Curr.Opin. Chem Biol, 2:512-518), the RU486-inducible system (Wang et al.,1997 Nat. Biotech., 15:239-243 and Wang et al., 1997 Gene Ther.,4:432-441) and the rapamycin-inducible system (Magari et al., 1997 J.Clin. Invest., 100: 2865-2872). According to an embodiment of theinvention, the promoter is an arabinose inducible promoter.

Any suitable bacterial host cell is contemplated for use in the presentinvention as would be understood by a person skilled in the art basedupon the disclosure provided herein. For example, suitable bacteria forthis purpose include Escherichia, Enterobacter, Azotobacter, Erwinia,Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia,Shigella, Rhizobia, Vitreoscilla, Paracoccus, or a combination thereof,without limitation. Any suitable strain of any such suitable bacteria isalso contemplated by the present invention. Further, the use of suitablemutated cells, as would be recognized by a person of skill in the art,is also contemplated by the present invention. A person of skill in theart would readily be able to select an appropriate host cell to useunder specific circumstances based upon the guidance provided herein.

Examples of suitable inducible E. coli expression vectors include,without limitation, pTrc (Amann et al., 1988 Gene, 69:301-315), thearabinose expression vectors (for example, Pbad18, Guzman et al., 1995J. Bacteriol., 177:4121-4130), and pETlid (Studier et al., 1990 Methodsin Enzymology, 185:60-89). Target gene expression from the pTrc vectorrelies on host RNA polymerase transcription from a hybrid trp-lac fusionpromoter. Target gene expression from the pETlid vector relies ontranscription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase T7 gn 1. This viral polymerase issupplied by host strains BL21 (DE3) or HMS I 74(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV5 promoter. The P_(BAD) system relies on the induciblearabinose promoter that is regulated by the araC gene. The promoter isinduced in the presence of arabinose.

Other embodiments of the present invention utilize arabinose-regulatedexpression vectors, or vectors where the expression of the recombinantprotein of interest is under the control of an arabinose promoter, forexample, the promoter for the E. coli arabinose operon, PBAD or PARA,without limitation.

A nucleic acid (nucleotide) sequence encoding any desired protein iscontemplated by the present invention. The nucleotide sequence may be afull or partial naturally-occurring nucleotide sequence or a full orpartial altered nucleotide sequence, or any sequence that hybridizesthereto under stringent conditions. References herein to nucleic acidsequences that correspond to a gene refer to any nucleic acid sequenceexpressible as the desired protein.

For example, such altered nucleic acid sequences include one nucleotidedeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleotidealterations is determined by multiplying the total number of nucleotidesin any sequence by the numerical percent of the respective percentidentity (divided by 100) and subtracting that product from said totalnumber of nucleotides in said sequence.

For instance, the present invention contemplates use of a nucleotidesequence that has at least 70% identity to a certain nucleic acidsequence; a degenerate variant thereof or a fragment thereof, whereinthe sequence may include up to n_(n) nucleic acid alterations over theentire polynucleotide region of the nucleic acid sequence, wherein n_(n)is the maximum number of alterations and is calculated by the formula:

n _(n) =x _(n)−(x _(n) ·y),

in which x_(n) is the total number of nucleic acids of any sequence andy has a value of 0.70, wherein any non-integer product of x_(n) and y isrounded down to the nearest integer prior to subtracting such productfrom x_(n). Of course, y may also have a value of 0.80 for 80%, 0.85 for85%, 0.90 for 90%, 0.94 for 94%, 0.95 for 95%, 0.96 for 96%, 0.97 for97%, 0.98 for 98%, or 0.99 for 99%, etc. Alterations of a sequence maycreate nonsense, missense or frameshift mutations in this codingsequence and thereby alter the polypeptide encoded by the polynucleotidefollowing such alterations.

The present invention contemplates the use of degenerate variants, or afragment thereof. As defined herein, a “degenerate variant” is apolynucleotide that differs from the nucleotide sequence (and fragmentsthereof) due to degeneracy of the genetic code, but still encodes thesame protein.

The nucleic acid may comprise DNA, chromosomal DNA, cDNA and RNA and mayfurther comprise heterologous nucleotides. In accordance with variousembodiments, the nucleic acid hybridizes to a certain nucleic acid, acomplement thereof, a degenerate variant thereof, or a fragment thereof,under high stringency hybridization conditions. In yet otherembodiments, the polynucleotide hybridizes under intermediate stringencyhybridization conditions.

It will be appreciated that the nucleic acids may be obtained fromnatural, synthetic or semi-synthetic sources; furthermore, thenucleotide sequence may be a naturally occurring sequence, or it may berelated by mutation, including single or multiple base substitutions,deletions, insertions and inversions, to such a naturally occurringsequence. The nucleic acid molecule may be RNA, DNA, single stranded ordouble stranded, linear or covalently closed circular form.

Examples of stringency conditions are shown in the Stringency ConditionsTable below: highly stringent conditions are those that are at least asstringent as, for example, conditions A-F; stringent conditions are atleast as stringent as, for example, conditions G-L; and reducedstringency conditions are at least as stringent as, for example,conditions M-R.

STRINGENCY CONDITIONS Poly- Hybrid Hybridization Wash Stringencynucleotide Length Temperature and Temperature Condition Hybrid (bp)^(I)Buffer^(H) and Buffer^(H) A DNA:DNA >50 65EC; 1xSSC -or- 65EC; 42EC;1xSSC, 50% 0.3xSSC formamide B DNA:DNA <50 T_(B); 1xSSC T_(B); 1xSSC CDNA:RNA >50 67EC; 1xSSC -or- 67EC; 45EC; 1xSSC, 50% 0.3xSSC formamide DDNA:RNA <50 T_(D); 1xSSC T_(D); 1xSSC E RNA:RNA >50 70EC; 1xSSC -or-70EC; 50EC; 1xSSC, 50% 0.3xSSC formamide F RNA:RNA <50 T_(F); 1xSSCT_(f); 1xSSC G DNA:DNA >50 65EC; 4xSSC -or- 65EC; 1xSSC 42EC; 4xSSC, 50%formamide H DNA:DNA <50 T_(H); 4xSSC T_(H); 4xSSC I DNA:RNA >50 67EC;4xSSC -or- 67EC; 1xSSC 45EC; 4xSSC, 50% formamide J DNA:RNA <50 T_(J);4xSSC T_(J); 4xSSC K RNA:RNA >50 70EC; 4xSSC -or- 67EC; 1xSSC 50EC;4xSSC, 50% formamide L RNA:RNA <50 T_(L); 2xSSC T_(L); 2xSSC MDNA:DNA >50 50EC; 4xSSC -or- 50EC; 2xSSC 40EC; 6xSSC, 50% formamide NDNA:DNA <50 T_(N); 6xSSC T_(N); 6xSSC O DNA:RNA >50 55EC; 4xSSC -or-55EC; 2xSSC 42EC; 6xSSC, 50% formamide P DNA:RNA <50 T_(P); 6xSSC T_(P);6xSSC Q RNA:RNA >50 60EC; 4xSSC -or- 60EC; 2xSSC 45EC; 6xSSC, 50%formamide R RNA:RNA <50 T_(R); 4xSSC T_(R); 4xSSC bp^(I): The hybridlength is that anticipated for the hybridized region(s) of thehybridizing polynucleotides. When hybridizing a polynucleotide to atarget polynucleotide of unknown sequence, the hybrid length is assumedto be that of the hybridizing polynucleotide. When polynucleotides ofknown sequence are hybridized, the hybrid length can be determined byaligning the sequences of the polynucleotides and identifying the regionor regions of optimal sequence complementarities. Buffer^(H): SSPE(1xSSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can besubstituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) inthe hybridization and wash buffers; washes are performed for 15 minutesafter hybridization is complete. T_(B) through T_(R): The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10EC less than the melting temperature (T_(m)) of thehybrid, where T_(m) is determined according to the following equations.For hybrids less than 18 base pairs in length, T_(m)(EC) = 2(# of A + Tbases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairsin length, T_(m)(EC) = 81.5 + 16.6(log₁₀[Na⁺]) + 0.41(% G + C) −(600/N), where N is the number of bases in the hybrid, and ([Na⁺] is theconcentration of sodium ions in the hybridization buffer ([Na⁺] for1xSSC = 0.165 M).

Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference.

The invention contemplates using polynucleotides that are fullycomplementary to these polynucleotides as well as antisense sequences.The antisense sequences, also referred to as antisense oligonucleotides,include both internally generated and externally administered sequencesthat block expression of polynucleotides encoding the polypeptides ofthe invention. The antisense sequences of the invention comprise, forexample, about 15-20 base pairs, without limitation. The antisensesequences can be designed, for example, to inhibit transcription bypreventing promoter binding to an upstream nontranslated sequence or bypreventing translation of a transcript encoding a polypeptide of theinvention by preventing the ribosome from binding.

The polynucleotides may be prepared or obtained in any suitable manner(for example, by chemical synthesis, from DNA libraries, from theorganism itself) and can take various forms (such as, single-stranded,double-stranded, vectors, probes, primers) as would be understood bypersons of skill in the art. The term “polynucleotide” includes DNA andRNA, and also their analogs, such as those containing modifiedbackbones. According to further implementations of the presentinvention, the polynucleotides comprise a DNA library, such as a cDNAlibrary. 2086 Protein Expression Systems:

A recombinant microorganism capable of expressing a Neisseriameningitidis serogroup B 2086 polypeptide is provided in accordance withan embodiment of the present invention. The recombinant microorganismcomprises an expression control sequence having promoter sequences andinitiator sequences, and a nucleotide sequence which codes for a 2086polypeptide, the nucleotide sequence being located 3′ to the promoterand initiator sequences. In a further aspect, there is provided a hostcell comprising a recombinant 2086 polynucleotide as described hereinand in WO 03/063766 and WO 04/094596, which are incorporated herein byreference in their entirety. As such the present invention provides amethod for producing a recombinant 2086 protein, as described in WO03/063766 and WO 04/094596, for example, without limitation.

Once host cells expressing a desired protein or polypeptide of theinvention have been constructed by transforming, transfecting orinfecting host cells with plasmids containing the corresponding 2086polynucleotide, the host cells are cultured under conditions such thatthe polypeptides are expressed in accordance with the methods of thepresent invention. The polypeptide may then be isolated substantiallyfree of contaminating host cell components by techniques well known tothose skilled in the art.

Threshold Parameters:

Several parameters may be used to monitor and control the progress ofthe culture in terms of cell growth and recombinant protein expression.Such parameters include, but are not limited to, optical density (OD),dissolved oxygen (DO), pH, nutrient/energy consumption (such as carbonsource), accumulation of metabolic by-products (for example, aceticacid), harvest time, and temperature. Any suitable parameter orcombination of parameters is contemplated for use in the presentinvention, as would be understood by a person of ordinary skill in theart, based upon the guidance provided herein.

A threshold parameter is established to determine the point at which theinducer is to be continuously added to the culture (that is, fed to theculture over time). The threshold parameter is a predeterminedparameter. An appropriate threshold parameter, such as a predeterminedoptical density, is readily determined by a person of skill in the artbased upon the guidance provided herein in accordance with variousembodiments of the present invention. One threshold parameter or acombination of threshold parameters may be used.

The parameter or combination of parameters may be monitored at anysuitable time intervals in the culture. For example, OD₆₀₀ and glucoseconcentrations may be monitored at one-hour, half-hour, or quarter-hourintervals, without limitation.

The optical density is used as the threshold parameter for initiation ofthe continuous inducer feed in accordance with an embodiment of thepresent invention. When the cell density of the culture achieves apredetermined threshold parameter, such as an optical density of about70 to about 110, the inducer is then fed to the culture as describedherein. A narrower range may be established for the threshold parameter.For instance, the present invention contemplates that one may initiatethe continuous addition of the inducer to the culture when cell densityof the culture achieves an optical density about 70 to about 105, about75 to about 100, about 75 to about 95, about 75 to about 85, about 76 toabout 84, about 78 to about 82, or about 80, in accordance withembodiments of the present invention.

The present invention also contemplates the use of threshold parametersto signal the initiation and/or termination of the feeding of the carbonsource.

Any suitable device or combination of devices is contemplated for use inmonitoring the threshold parameter(s), as would be known to persons ofskill in the art. For instance, a probe or combination of probes formeasuring a threshold parameter, may be mounted on the fermentationdevice (the “fermentor”) in any suitable manner, without limitation.

Constant Feed Rates:

The constant feed rates refer to the rate at which the inducer(s) and/orcarbon source(s) is added to the culture. The inducer is added to theculture after a threshold parameter has been achieved. The carbon sourcemay also be added to the culture after a threshold parameter is achieved(and likewise, addition of the carbon source may be terminated uponachieving a threshold parameter). These threshold parameters include,without limitation, optical density (OD), dissolved oxygen (DO), pH,concentration of nutrient in the culture medium, total concentration ofthe first carbon source added to the culture medium, or any combinationthereof.

Any suitable constant rate is used to continuously add the inducerand/or carbon source to the culture as would be understood by a personskilled in the art based upon the guidance provided herein. Inaccordance with various embodiments of the present invention, a suitableconstant rate is determined by DO-stat, as described in the examplesbelow. For example, a feed rate equivalent to the DO-stat controller maybe selected by adding enough glucose to bring the concentration up to 15and 24 g/L every hour, without limitation.

For example, the inducer and/or carbon source may be added to theculture at a constant rate until a certain amount of inducer and/orcarbon source, such as from about 4 g/L to about 40 g/L, such as, 4 g/L,5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 9.5 g/L, 9.75 g/L, 10 g/L, 10.25 g/L,10.5 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, 25 g/L, 26 g/L, 27g/L, 28 g/L, 29 g/L, 30 g/L, 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, based on the total volume of theculture, has been added to the culture, without limitation. According tovarious embodiments, the total amount of inducer and/or carbon sourcefed to the culture is about 5 g/L to about 20 g/L, 7 g/L to about 15g/L, 8 g/L to about 14 g/L, 9 g/L to about 11 g/L, or about 10 g/L.

The total amount of inducer to be added to the culture may be offset bythe total amount of carbon source added to the culture. For instance,when the carbon source is glucose and the inducer is arabinose, theamount of inducer added may be reduced by addition of glucose. Forexample, in an embodiment, a total of 10 g/L of an inducer (that is,such as arabinose is added to a culture and 11 g/L of a carbon source,such as glucose is added. The protein yield obtained in this mannerapproximates the yield when a total amount of 20 g/L of arabinose (thatis, 20,000 g or 20 kg total in a 1,000 L culture of arabinose) and noglucose is used. Thus, this offset provided by the present methods isadvantageous given the high cost of arabinose relative to glucose.

According to various embodiments of the invention, the constant rate atwhich the inducer and/or carbon source is added to the culture may beset in the range from about 1.5 g/L to about 24 g/L every hour. Forexample, where the carbon source is glucose, the constant rate for theaddition of glucose may include, without limitation, 1.8 g glucose/L/h,3.3 g glucose/L/h, 6.7 g glucose/L/h, 15 g glucose/L/h, 16.4 gglucose/L/h, 18 g glucose/L/h, 24 g glucose/L/h, etc. According tovarious embodiments, an inducer such as arabinose is added at constantrates of about 1.5 g/L/h to about 16 g/L/h.

According to various embodiments, once a threshold parameter has beenachieved, the feed on the carbon source may be continued, stopped ortemporarily interrupted. The feed of the carbon source may beinterrupted in which case the feed will re-start at the constant rateonce a threshold for starting the feed has been achieved. Thus,according to an embodiment, a start threshold and a stop threshold maybe used to regulate the feed of the carbon source into the culture.According to another embodiment, both glucose and arabinose are fed at aconstant rate based on the threshold parameter, without stopping orrestarting the feed.

The appropriate total amount of carbon source to add to any specificculture can be readily determined by a person of skill in the art basedupon the guidance provided herein. The total amount of carbon sourceadded to the culture may range from about 1 g/L to about 100 g/L (basedon the total volume in liters of the culture) according to an embodimentof the present invention. For example, according to an embodiment, 50g/L of glucose is added during the growth phase by starting with 10 g/Lin the medium, commencing the constant rate glucose feed when theglucose level reaches zero, and continuing the constant rate glucosefeed until OD achieves 80 at which time about 40 g/L of glucose willhave been fed in addition to the initial 10 g/L of glucose. According toan embodiment, the total amounts of carbon source are provided inconcentrated form for ease of scalability. These amounts are readilyconverted into the total mass of the carbon source to be used in aparticular circumstance. For example, when 10 g/L of carbon source is tobe added to a 1,000L culture, the total amount of carbon source to beadded is readily determined as 10 g/L×1,000 L=10,000 grams (or 10 kg) oftotal carbon source. The total amount of carbon source added may serveas a threshold parameter in accordance with various embodiments asdescribed herein.

The appropriate total amount of inducer to add to any specific culturemay be readily determined by a person of skill in the art based upon theguidance provided herein. The total amount of inducer added to theculture may range from about 4 g/L to about 40 g/L (based on the totalvolume in liters of the culture) in accordance with various embodiments.According to various embodiments, the total amount of carbon sourceadded to the culture is about 5 g/L to about 20 g/L, 7 g/L to about 15g/L, 8 g/L to about 14 g/L, 9 g/L to about 11 g/L, or about 10 g/L basedon the total volume of the culture. According to an embodiment, thetotal amounts of inducer are provided in concentrated form for ease ofscalability. These amounts are readily converted into the total mass ofthe inducer to be used in a particular circumstance. For example, when10 g/L of inducer is to be added to a 1,000 L culture, the total amountof inducer to be added is readily determined as 10 g/L×1,000 L=10,000grams (or 10 kg) of total inducer.

The fresh culture medium will typically contain an initial amount of afirst carbon source at the time of inoculation with a host cell, thuscreating a culture. This initial concentration may be monitored and theconcentration of the first carbon source used as a threshold parameter.

Any suitable supplement or nutrient besides a carbon source may also befed into the culture in appropriate amounts. The other nutrient orsupplement may be monitored and thresholds set appropriately.Supplements such as nitrogen or inorganic phosphate sources arecontemplated for use in the present invention. Non-limiting examples ofcompounds that are contemplated for use in the methods of the presentinvention include KH₂PO₄, K₂HPO₄, sodium citrate, dihydrate, (NH₄)₂SO₄,MgSO₄, (Na)₂SO₄, CaCl₂, FeSO₄, chloramphenicol or any combinationthereof. The use of an additional carbon source or sources is alsocontemplated.

Optical Density and Log Growth Phase:

The introduction of a bacterial host cell to fresh culture media createsa culture that typically goes through four more-or-less distinct phasesof growth: (i) lag phase, (ii) log (logarithmic or exponential) phase,(iii) stationary phase, and (iv) decline (death) phase. The log phaseitself may be further divided into various phases, such as early loggrowth phase, mid log growth phase, and late log growth phase. Opticaldensity is related to the phase of log growth. Log growth phase andoptical density may also be used as threshold parameters to signal thestart and/or stop of the constant feed of the carbon source and/orinducer.

For example, induction, or continuous addition of the inducer maycommence at early-log growth phase, mid-log growth phase, and late-loggrowth phase. Late log growth phase may occur at an OD of about 70 toabout 110. In an embodiment of the invention, the constant rate feed ofthe inducer will start in the late-log growth phase of the culturemedium or at an OD of about 70 to about 110, about 70 to about 105,about 75 to about 85, or about 80 in accordance with variousembodiments.

OD may be measured at various wavelengths that are commonly employed bythose of skill in the art. Typically, OD₆₀₀ is used as a measure of cellgrowth and density of cells in the culture. Unless otherwise indicated,“OD” as used herein refers to OD₆₀₀.

Dissolved Oxygen:

Another parameter that may serve as a trigger for the start and/or stopof the feed controller is dissolved oxygen (DO) (that is, DO-stat fedbatch fermentation). DO may be controlled by adjusting the agitation,airflow, oxygen supplement, and pressure in the vessel to contain theculture media. The threshold of DO may be set in the range from 5% to80% DO, such as, 20%, 40%, or 80%. Once a threshold has been met, thefeed controller for a carbon source or inducer may be turned on untilthe threshold has been met that signals the stop of the control feed.The stop threshold may be another DO threshold or another parameter,such as the amount of the carbon source or inducer. For example,whenever the DO rises above 30% or 40% in a culture medium, the feedcontroller may start until which time that the DO falls to 20%, oralternatively, until 0.5 g/L or 1 g/L of a carbon source or inducer hasbeen newly added in accordance with various embodiments of the presentinvention.

pH:

Another parameter that may serve as a trigger for the start and/or stopof the feed controller is pH (that is, pH-stat fed-batch fermentation).pH may be controlled by addition of base or acid to culture media. Thethreshold of pH may be set in the range from 6.8 to 7.2, such as 7.0.Once a threshold has been met, the feed controller for a carbon sourceor inducer may be turned on until the threshold has been met thatsignals the stop of the control feed. The stop threshold may be anotherpH threshold or another parameter, such as the amount of the carbonsource or inducer. For example, whenever the pH rises to 6.97 in aculture medium, the feed controller may start until which time that thepH falls to 6.95, or alternatively, until 1 g/L of a carbon source orinducer has been newly added in accordance with various embodiments ofthe present invention.

Harvest Time:

Harvest time represents the amount of time that passes after the initialinduction or addition of an inducer. Any suitable harvest time iscontemplated by the present invention. Harvest time may range from about2 hours to about 10 hours, about 2 hours to about 8 hours, about 2.5hours to about 7 hours, about 3 hours to about 6 hours, etc., inaccordance with various embodiments of the present invention. Using theconstant feed rate, harvest time, and total amount of inducer, those ofordinary skill in the art will appreciate how each parameter may beadjusted to achieve the desired results. Persons skilled in the artwould understand when to harvest based on the amount of arabinose fedbecause they could readily determine the amount fed based on the feedrate and the time period. In this manner, final concentrations of theinducer of 5, 10, 20, 30, and 40 g/L fed in 3 hours may be achieved, forexample, without limitation.

Concentration of the Inducer:

An inducer in any suitable concentration is contemplated by the presentinvention. Concentrations of the inducer useful to induce host cells mayrange from about 0.00001% to about 20% (v/v), without limitations.

Temperature:

The culture of the present embodiments may be incubated at anytemperature that permits growth of the cells. Various temperatures atwhich to incubate the culture associated with abundant growth include,without limitation, 22° C., 28° C., 37° C., or any combination thereof.

Fermentation Device

Any suitable fermentation device (that is, “fermentor”) is contemplatedfor use in the present invention, as would be known to persons of skillin the art. For example, the fermentor may contain any number ofimpellers (such as, Rushton impellers), intakes and/or measurementprobes. In accordance with an embodiment, the fermentor is configured toinclude three Rushton impellers and a ring or tube sparger forintroduction of air into the fermentor. The present inventioncontemplates the use of manual and/or computer-based systems. As such,the fermentation system may interface with a computerized system formonitoring and control of fermentations. In this manner, the system maybe fully or partially automated, in accordance with embodiments of thepresent invention.

Compositions of the Present Invention:

Compositions comprising recombinant proteins, such as those prepared inaccordance with the methods of the present invention are providedherein, in accordance with embodiments of the present invention.Compositions of the present invention comprise recombinant protein inhigh density in a culture, such as the recombinant proteins prepared inaccordance with the methods of the present invention, without intendingto be limited thereto.

The composition comprises a culture having recombinant protein at adensity of at least about 1.5 g/L based on the total volume of theculture. The density of the recombinant protein is at least about 1.7g/L based on the total volume of the culture according to a furtherembodiment of the present invention. The density of the recombinantprotein is at least about 2.0 g/L based on the total volume of theculture according to another embodiment of the present invention. Thedensity of the recombinant protein is at least about 3.0 g/L based onthe total volume of the culture according to another embodiment of thepresent invention.

A composition comprising recombinant 2086 protein is provided in anembodiment of the present invention. The 2086 protein, as referred toherein, is a protein expressed by a polynucleotide that corresponds tothe 2086 gene in N. meningitidis serogroup B, including any fragment,derivatives or mutations thereof. Non-limiting exemplary 2086 proteinsand polynucleotides are described in WO 03/063766 and WO 04/094596.

The recombinant 2086 protein composition comprises recombinant 2086protein in a culture wherein the recombinant 2086 protein is at adensity of at least about 1.5 g/L based on the total volume of theculture. The density of the recombinant 2086 protein is at least about1.7 g/L based on the total volume of the culture according to a furtherembodiment of the present invention. The density of the recombinant 2086protein is at least about 2.0 g/L based on the total volume of theculture according to another embodiment of the present invention. Thedensity of the recombinant 2086 protein is at least about 3.0 g/L basedon the total volume of the culture according to another embodiment ofthe present invention.

The compositions of the present invention may comprise any protein, suchas a protein prepared in accordance with a method of the presentinvention. The recombinant proteins may be lipidated or nonlipidatedproteins. In an embodiment of the invention, the recombinant protein isrecombinant 2086 protein either lipidated or non-lipidated. Therecombinant 2086 protein may be a 2086 subfamily A or subfamily Bprotein, or a combination thereof. The compositions of the presentinvention may include one protein or more than one protein. The proteinsmay be related or unrelated proteins. For example, a composition of thepresent invention may include 2086 protein corresponding to one or morestrains of subfamily A and/or one or more strains of subfamily B.

Compositions comprising material for use in conducting the methods ofthe present invention are also provided herein. Such compositionsinclude the necessary components for the culture, including recombinantcells and nutrients, in accordance with embodiments of the presentinvention. The various compositions may be provided together in a kit,in accordance with an embodiment of the present invention. For example,the components to form the culture may be conveniently pre-packaged inthe required amounts to facilitate use in laboratory or industrialsettings, without limitation. Such a kit may also include labels,indicia and directions to facilitate the use of each component and themanner of combining the components in accordance with variousembodiments of the present invention.

The following examples are included to demonstrate various embodimentsof the invention. It should be appreciated by those skilled in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute various modesfor its practice. However, those of skill in the art should, in view ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLES Example 1 Fed-Batch Fermentation with Constant Rate Feed

E. coli (pPW62) subfamily B was used as a model strain for a fed-batchfermentation process. Based on the results, the process will be appliedto the subfamily A E. coli (pPW102).

A medium and feed solution for the fed-batch fermentation was preparedusing the components as listed in the following tables.

Medium and feed solution

TABLE 1 Basal Medium Component Quantity per Liter Dextrose, Anhydrous 10g KH₂PO₄ 3 g K₂HPO₄ 7 g (NH₄)₂SO₄ 1 g Sodium Citrate, Dihydrate 1 gMgSO₄•7H₂O 1 g (Na)₂SO₄ 0.58 g CaCl₂•2H₂O 0.075 g FeSO₄•7H₂O 0.09 g1000x Trace Metal Stock Solution (Table 1 mL 6) Chloramphenicol 15 mg

TABLE 2 1000x Trace Metal Stock Solution Component Quantity per LiterZnSO₄•7H₂O  30 g CuSO₄•5H₂O   9 g MnSO₄•H₂O 4.2 g CoCl₂•6H₂O 0.6 gMolybdic Acid, Ammonium Salt, 1.5 g Tetrahydrate

TABLE 3 Glucose Concentrate Feed Solution Component Quantity per LiterGlucose 500/700 g KH₂PO₄ 3 g K₂HPO₄ 5 g (NH₄)₂SO₄ 2 g

TABLE 4 Arabinose Concentrate Feed Solution Component Quantity per LiterArabinose 250/500 g and varied based on experiment KH₂PO₄ 3 g K₂HPO₄ 5 g(NH₄)₂SO₄ 2 g

Methods:

Fed-batch fermentation with constant rate feed was used to achieve highcell density in E. coli fermentation. The initial glucose concentrationwas 10 g/L in the medium. The (NH₄)₂SO₄ concentration was increased to 3g/L in the fermentation medium, but held at 1 g/L in the seed culturemedium. To determine the feed rate, DO-stat fed-batch was performedfirst. The DO level was controlled at 20% by a cascade controller thatincreased the agitation speed to maximum and then used oxygensupplementation. When glucose was depleted, the DO rose sharply (above40%) and glucose concentrate was added to final concentration of 1 g/Lin the fermentor. After each addition of glucose, the pump remained offfor a set time before being allowed to make the next addition. Themaximum OD of about 160 was achieved when DO-stat fed-batch fermentationwas performed. The constant rate was then chosen to be equivalent to theDO-stat controller adding enough glucose to bring the concentration upto 18 g/L or 24 g/L every hour. During the fed-batch fermentation withconstant rate feed, the glucose feed was turned on at the desiredconstant rate when there was a sharp rise to 40% in DO.

The seed cultures were started using one vial of E. coli (pPW62) perliter of basal medium +15 μg/mL chloramphenicol in a 2800 mL Fernbachflask. The flasks were incubated at 32° C., 150 rpm overnight (˜16 hrs).The final OD₆₀₀ was normally ˜3. 10% inoculum size was used to inoculateeach fermentor. Each fermentor used 3 Rushton impellers and a ringsparger. Initial setpoints: temp: 36° C., pH: 7.00±0.05 (controlled with7.4 N NH₄OH), airflow: ˜1 vwm, DO: 20%. The DO was controlled by acascade of agitation (min: 150 rpm, max: 1000 rpm) and O₂ addition via agas mix unit. Foam was controlled, if needed, by manual addition ofPPG-2000. 0.35 mL/L AF was added to the medium before sterilization.During the fermentation, samples were taken hourly to monitor glucose,pH, and OD₆₀₀ off-line. Supernatants were prepared from 1 mL samples andstored for later analysis of organic acids by HPLC.

Results: Fed-Batch Fermentation with Constant Rate Feed:

FIG. 1 shows the time courses of OD, glucose consumption, and aceticacid accumulation with constant feed rates. Maximum ODs of 158 and 150were obtained with constant feed rates of 24 g glucose/L/h and 18 gglucose/L/h, respectively. High glucose 28 g/L was accumulated duringthe run when a feed rate of 24 g/L/h was used. Glucose accumulates to 12g/L when a 18 g/L/h feed rate was used. Little acetic acid (that is,less than 1.5 g/L) was produced in both cases. The exponential growthphase ended close to OD 100. The specific growth rate was approximately0.60 (hr⁻¹) in both cases.

To reduce the glucose accumulation, low constant feed rates of 16.4g/L/h and 15 g/L/h were examined. FIG. 2 shows the time courses of OD,glucose consumption, and acetic acid accumulation with constant feedrates mentioned above. Maximum ODs were 142 and 147, respectively. As inprevious runs, the culture with the faster feed rate accumulated moreglucose, although the amount of glucose accumulated was much less thanin previous experiments. About 8 g/L of glucose was accumulated duringthe 16.4 g/L/h, and 5.4 g/L of glucose during the 15 g/L/h fermentation.Little acetic acid (such as less than 1.5 g/L) was produced in bothcases (see FIG. 2). The specific growth rate was approximately 0.60(hr⁻¹) in both cases. Thus, the specific growth rate was not affected bythe feed rate between 15 g/L/h and 24 g/L/h.

Induction at Various Growth ODs:

A 15 g glucose/L/h constant feed rate was used for the arabinoseinduction study because it resulted in high cell density and low glucoseand acetic acid accumulation. In this experiment, inductions at mid-loggrowth phase OD ˜55 and late-log growth phase OD ˜80 were compared. Theculture was induced by simply substituting the arabinose feed for theglucose feed and feeding arabinose at a constant rate of 13.4 g/L/h. Atotal of 40 g/L arbinose was added to each culture over the course of 3hours. After induction, samples were taken every hour for rLP2086 assayby SDS-PAGE, organic acid and arabinose assays by HPLC.

FIG. 3 shows the time courses of OD and rLP2086 production when inducedat ODs ˜55 and ˜80. Both maximum OD and rLP2086 yield were higher whencells were induced at OD ˜80 (maximum OD: 101 vs. 84; maximum yield: 1.8g/L vs. 1.2 g/L).

Induction with Various Arabinose Levels

The purpose of the following experiments was to assess the total amountof arabinose fed to the culture and examine whether reduction of thetotal amount of arabinose fed to the culture would still result in highrLP2086 expression. Arabinose concentrate was fed to 4 differentcultures each at different feed rates over the course of 3 hours,resulting in final arabinose concentrations of 10, 20, 30, and 40 g/L.All cultures were induced at OD₆₀₀˜80. FIG. 4 shows the time courses ofOD and rLP2086 production. Table 5 summarizes the OD and rLP2086 yieldsfor each of the four conditions. It shows the maximum rLP2086 yields of:1.2 g/L for the 10 g/L total arabinose added; 1.6 g/L for the 20 g/Ltotal arabinose added; 1.7 g/L for the 30 g/L total arabinose added; 2.0g/L for the 40 g/L total arabinose added. An arabinose feed of between20 g/L and 40 g/L resulted in similar rLP2086 yield, however, 10 g/Larabinose produced much less rLP2086 (that is, 1.2 g/L). These resultssuggest that the total amount of arabinose added for induction can bereduced from 40 g/L to 20 g/L without reducing the rLP2086 productivity.Thus, the reduction in arabinose usage would be more cost effective,especially considering the high cost of arabinose (approximately $500/kgUS).

TABLE 5 Induction with various arabinose levels Lot Total arabinose fed(g/L) Maximum rLP2086 (g/L) X-BRN05-027 10 1.2 X-BRN05-024 20 1.6X-BRN05-025 30 1.7 X-BRN10-114 40 2.0

Arabinose Addition Method Comparison:

The following experiment was conducted to examine whether a continuousfeeding strategy is superior to a simple batch addition strategy whenapplied to arabinose for induction. In run X-BRN05-039, 20 g/L ofarabinose was added to the fermentor all at once, rather than feedingover the course of time, when the OD was about 80. FIG. 5 shows the timecourses of OD, glucose and arabinose consumption, and rLP2086production. A maximum of 1.3 g/L of rLP2086 was obtained. Batch additionof the arabinose, although operationally simpler, produced less rLP2086than continuous feeding. Thus, a continuous arabinose feed strategy issuperior to simple batch addition.

To examine whether the arabinose can be more efficiently used byreducing the arabinose feed rate, feed rates of 3.3 g arabinose/L/h and6.7 g arabinose/L/h were compared. FIG. 6 shows the time courses of ODand rLP2086 production. Arabinose concentrate was fed to one culture ata feed rate of 6.7 g/L/h over the course of 3 hours, and a secondculture was fed at a rate of 3.3 g/L/h over the course of 6 hours. Forboth cultures, the total amount of arabinose added was 20 g/L. As shownin FIG. 6, both conditions produced the same amount of maximum rLP2086(that is, 2.2 g/L), but there were differences in the kinetics of theproduction. The higher feed rate resulted in a higher production rate.Maximum rLP2086 was achieved at 3 hours and 6 hours after induction withfeed rates of 6.7 g/L/h and 3.3 g/L/h, respectively. The advantage ofusing a higher feed rate (that is, 6.7 g/L/h) is that production cost(for example, utility cost) will be lower when using a higher feed ratethan when using a lower feed rate.

Effect of Induction Time on rLP2086 Expression Yield

To determine the optimum harvest time, the normal feeding profile (20g/L arabinose fed over 3 hours) was extended to 40 g/L fed over 6 hours.In runs X-BRN05-028 and X-BRN05-029, the cells were induced at OD ˜55and OD ˜80, respectively. FIG. 7 shows the time courses of OD andrLP2086 production. Although the arabinose feed extended from 3 hours to6 hours, interestingly, the peak titer was still obtained around 3 hoursafter induction. The product titer was slightly higher in the culturethat was induced at higher OD. The maximum rLP2086 yield at induction OD˜55 was 2.0 g/L (X-BRN05-028) while it was 2.4 g/L at induction OD˜80(X-BRN05-029). This result suggests that the cells should be harvested 3hours after induction.

Comparison of Feed Solution with and without Salts

To examine whether added salts are essential in the glucose andarabinose feed solutions, plain glucose and arabinose feeds werecompared with the standard glucose+salts (that is,K₂HPO₄/KH₂PO₄+(NH₄)₂SO₄) and arabinose+salts feeds. For both cultures,20 g/L of arabinose was fed over the course of 3 hours. The growth andrLP2086 production profiles were very similar. The maximum rLP2086 yieldwas 1.8 g/L when salts were added to the feeds, and 2.0 g/L when glucoseand arabinose feeds were prepared without salts. These results suggestthat there is no need to add salts to the glucose and arabinose feedsolutions.

Fed-Batch Fermentation with Constant Rate Feed for Subfamily B Strain:

Seed cultures were started by inoculating 1 L of basal medium containing15 μg/mL chloramphenicol with 1 ml of thawed working seed. The culturewas grown in a 2.8 L Fernbach flask and was incubated for about 16 hoursat 32° C. and 150 rpm. The final OD₆₀₀ was ˜3.0. 350 mL seed cultureswere aseptically transferred into 3.15 L basal medium containing 3 g/L(NH₄)₂SO₄ without chloramphenicol. The fermentation was controlled at pH7.0±0.05 by 7.4 N NH₄OH, temperature at 36° C., DO at 20%, and airflowat 1 vvm. The DO was controlled by a cascade of agitation (min: 150 rpm,max: 1000 rpm) and oxygen addition. Antifoam PPG-2000 was automaticallyadded to control foam. During the fermentation, samples were takenhourly to monitor glucose and OD off-line. After inoculation, the DOdropped from ˜100% to 20% and then was maintained at 20%. When there wasa sharp rise in DO from 20% to greater than 40% (usually 6 hours ElapsedFermentation Time (EFT)), the glucose (without salts) feed pump wasturned on at a rate of 15 g/L/h. As shown in FIG. 8, glucose wascompletely depleted by 6-hour EFT, resulting in a sharp rise in the DO.The samples were taken every half hour when OD reached ˜40. The glucosefeed was turned off at OD 90 and the arabinose feed was turned on at arate of 13.4 g/L/h. After 3 hours of arabinose (without salts) feeding(that is, a total 20 g/L arabinose addition), the arabinose feed wasturned off and the fermentation was allowed to continue for anotherhour. As shown in FIG. 8, an OD of 102 was obtained and 2.0 g/L of MnBrLP2086 was expressed based on SDS-PAGE (see FIG. 9). The peak occurred3 hours after induction (that is, ˜12-hour EFT). SDS-PAGE and WesternBlot showed that the expressed protein was indeed subfamily B rLP2086.

Application of Fed-Batch Fermentation Process for MnB rLP2086 SubfamilyA Strain:

To test whether the fed-batch fermentation process used for rLP2086subfamily B was applicable to rLP2086 subfamily A, the process wasconducted using the procedure established for the subfamily B strain.FIG. 10 shows the time courses of OD, glucose and arabinose consumption,and rLP2086 production. The growth and rLP2086 production profiles forsubfamily A were similar to those obtained for subfamily B (compare toFIG. 8). SDS-PAGE and Western Blot showed that expressed protein wasindeed subfamily A rLP2086 (see FIG. 11). Table 6 lists the maximum ODsand rLP2086 expression yields for six different subfamily A runs. Therange of maximum rLP2086 expression yield was 1.5-2.1 g/L (averagemaximum yield: 1.8±0.2 g/L), similar to the results from the fed-batchfermentations used to produce rLP2086 subfamily B. Thus, the fed-batchfermentation developed for subfamily B strain is also suitable forsubfamily A strain.

TABLE 6 Maximum subfamily A rLP2086 expression yield and OD Lot MaximumrLP2086 (g/L) Maximum OD X-BRN10-118 1.9 104 X-BRN10-119 1.9 104X-BRN10-120 1.6 100 X-BRN05-042 1.5 100 X-BRN05-043 2.0 77 X-BRN10-1212.1 92

Dual Glucose and Arabinose Feed during the Arabinose Induction

To reduce the amount of arabinose needed without reducing rLP2086production, dual glucose and arabinose feed during the induction periodwas investigated. The strategy was to feed 10 g/L of arabinose over 3hrs (half the usual amount) while continuing to feed glucose during theinduction phase at 25% (3.75 g/L/h), 50% (7.5 g/L/h), and 100% (15g/L/h) of the standard glucose feed rate. All of the feeds, glucose andarabinose, were prepared without additives.

FIGS. 12 a, 12 b, and 12 c, show the time course of subfamily B cellgrowth, the glucose, arabinose, acetic acid concentrations, and rLP2086production. All three runs were induced at OD ˜80. As shown in FIG. 12a, the OD of the 100% glucose feed run continued to rise afterinduction, peaking at 117, while the 50% run peaked at 106 (FIG. 12 b)and the 25% run held around 100 (FIG. 12 c) after induction. The 100%glucose feed began to accumulate glucose and arabinose by 3 hourspost-induction. The 50% glucose run only showed a slight amount ofglucose in the last sample (reading=0.21 g/L). There was no glucoseaccumulation in the 25% glucose run. None of three runs accumulated anyarabinose. The 100% (FIG. 12 a) and 50% (FIG. 12 b) glucose fed runsproduced ˜1.5 and 1.7 g/L rLP2086, respectively, while the 25% (FIG. 12c) run produced over 2.1 g/L. The 100% runs' production peaked beforethe arabinose ran out, suggesting that rLP2086 expression may have beensuppressed by the accumulation of glucose and acetic acid. The 50% runs'production peaked about the time the arabinose ran out and the 25% runs'production peaked after the arabinose ran out suggesting that 2086expression might not be suppressed as long as glucose concentration wascontrolled at minimal level (no glucose accumulated in FIGS. 12 b and 12c). Although only 10 g/L of arabinose was fed to these cultures, theirrLP2086 production was similar to that obtained when 20 g/L was fed.Simultaneous feeds of glucose and arabinose can reduce the arabinoseconsumption by 50% and still achieve the same rLP2086 yield when glucoseconcentration is controlled at a low level during the induction. Thus,the cost of chemicals can be reduced significantly.

To examine whether one can further reduce arabinose consumption andincrease rLP2086 yield, various glucose feed rates, total amounts ofarabinose fed, and induction ODs were investigated. Table 7 listsdifferent combinations of these conditions. It appears that glucose feedrate between 2.25 and 7.5 g/L/h and arabinose feed rate between 1.7 and6.7 g/L/h would not affect rLP2086 yield significantly. Induction ODsbetween 80 and 105 result in similar rLP2086 yield.

TABLE 7 Maximum OD and rLP2086 under different glucose feed rate,different amount of arabinose addition, and induction at various ODGlucose feed Arabinose Amount of Max- Maximum Lot rate feed ratearabinose Induction imum rLP2086 number (g/L/h) (g/L/h) fed OD OD (g/L)X- 3.75 1.7 5 g/L in 74 86 1.6 BRN10- 3 hours 127 X- 3.75 3.3 20 g/L in79 122 2.8 BRN05- 3 hours 056 X- 3.75 1.7 10. g/L in 94 122 3.0 BRN05- 6hours 058 X- 3.75 6.7 20. g/L in 110 124 2.7 BRN10- 3 hours 129 X- 3.753.3 20. g/L in 105 126 2.9 BRN05- 6 hours 059 X- 5.25 3.3 20. g/L in 102112 2.6 BRN05- 6 hours 061 X- 2.25 3.3 20. g/L in 93 108 2.4 BRN10- 6hours 130

Example 3 Scaled-Up Fed-Batch Fermentation to 100 L Scale

The seed culture was started by inoculating 2×1 L basal mediumcontaining 15 μg/mL chloramphenicol with 1 ml (that is, 1 vial) ofthawed working seed. The culture in 2.8 L Fernbach was incubated forabout 16 hours at 32° C. and 150 rpm in a rotary shaker.

Two 1 L overnight Fernbach seed cultures were aseptically transferredinto a 150 L termentor containing 70 L basal medium withoutchloramphenicol. The 150 L fermentation in basal medium was controlledat pH 7.0±0.05 by 7.4 N NH₄OH, temperature 36° C., DO 20%, and air flowat 1 vvm. The DO was controlled by a cascade of agitation and oxygenaddition. Antifoam PPG-2000 was automatically added to control foam.During the fermentation, the DO dropped from 100% to 20% and wasmaintained at 20%. When there was a sharp rise in DO from 20% to greaterthan 40% (usually at OD ˜20), signaling the depletion of glucose, thefeed pump was activated to deliver the glucose concentrate (that is, 500g/L) at a rate of 15 g glucose/L broth/h. During the fermentation,samples were taken hourly to monitor glucose and OD off-line. Thesamples were taken every half hour when OD reached ˜40. Once OD reached˜80, the glucose feed was stopped and the arabinose feed was started(for example, 500 g/L of arabinose concentrate) at a rate of 6.7 garabinose/L broth/h for 3 hours.

FIG. 13 a shows the time courses of subfamily B cell growth, the glucoseconsumption, acetic acid accumulation, and rLP2086 production at 100 Lscale. The fermentation profile at the 100 L scale was similar to thatseen at the small-scale. A maximum OD of 99 and a maximum yield of 1.9g/L rLP2086 were obtained. These results demonstrate that the fed-batchfermentation is scalable.

FIG. 13 b shows the time courses of subfamily A cell growth, the glucoseconsumption, acetic acid accumulation, and rLP2086 production at the 100L scale. The fermentation profile at 100 L scale was similar to thatseen at the small-scale. A maximum OD of 96 and a maximum yield of 2.0g/L rLP2086 were obtained. These results demonstrate that the fed-batchfermentation is scalable and robust.

FIG. 14 a shows the time courses of subfamily B cell density, glucose,arabinose, acetic acid concentrations, and rLP2086 yield with dualglucose and arabinose feed at 100 L scale. During the induction, feedrates were controlled at 3.75 and 1.67 g/L/h for glucose and arabinosefeeding, respectively. Dual arabinose and glucose was fed in 5 hours. Amaximum OD of 90 and a maximum yield of 1.8 g/L rLP2086 were obtained.The maximum rLP2086 yield appeared at 4-hour induction. Average maximumOD and average maximum rLP2086 yield for subfamily B were 84.8±6.8 and1.6±0.3 g/L, respectively. These results demonstrate that the fed-batchfermentation with dual glucose and arabinose feed during the inductionphase is scalable.

FIG. 14 b shows the time courses of subfamily A cell growth, the glucoseconsumption, acetic acid accumulation, and rLP2086 production at the 100L scale. The fermentation ran at the same conditions as in paragraph[0129] and cells were induced for 6 hours. A maximum OD of 89 and amaximum yield of 1.8 g/L rLP2086 were obtained. The maximum rLP2086yield appeared at 4-hour induction. Average maximum OD is 87.9±10.5 andaverage maximum rLP2086 yield is 1.8±0.2 g/L for subfamily A. Theseresults demonstrate that the fed-batch fermentation is scalable androbust.

While the invention has been described with reference to variousembodiments and examples, those skilled in the art recognize thatvarious modifications may be made to the invention without departingfrom the spirit and scope thereof.

1. A method for producing a recombinant protein comprising: culturing a recombinant bacterial cell to express a recombinant protein comprising continuously adding a carbon source to a culture comprising the recombinant bacterial cell and continuously adding an inducer to the culture after the culture achieves a threshold parameter, and isolating the recombinant protein from the culture.
 2. The method of claim 1, wherein the threshold parameter is optical density (OD), dissolved oxygen (DO), concentration of nutrient in culture medium, total concentration of carbon source added to culture medium, or any combination thereof.
 3. The method of claim 2, wherein the threshold parameter is optical density (OD) of the culture.
 4. The method of claim 3, comprising continuously adding the inducer to the culture when the cell density of the culture achieves an OD₆₀₀ of about 70 to about
 110. 5. The method of claim 4, comprising continuously adding the inducer to the culture when the cell density of the culture achieves an OD₆₀₀ of about 70 to about
 105. 6. The method of claim 5, comprising continuously adding the inducer to the culture when the cell density of the culture achieves an OD₆₀₀ of about 75 to about
 85. 7. The method of claim 6, comprising continuously adding the inducer to the culture when cell density of the culture achieves an OD₆₀₀ of about
 80. 8. The method of claim 1, comprising adding the inducer to the culture at a constant rate, adding the inducer to the culture by DO-stat feed or adding the inducer to the culture by pH-stat feed.
 9. The method of claim 1, comprising adding the inducer to the culture at a constant rate of about 3.35 g/L/h to about 16 g/L/h, adding the inducer to the culture by DO-stat feed or by pH-stat feed.
 10. The method of claim 1, wherein the total amount of the inducer added to the culture is about 4 g/L to about 40 g/L.
 11. The method of claim 10, wherein the total amount of the inducer added to the culture during inducer feed is from about 5 g/L to about 20 g/L.
 12. The method of claim 11, wherein the total amount of the inducer added to the culture during inducer feed is from about 7 g/L to about 15 g/L.
 13. The method of claim 12, wherein the total amount of the inducer added to the culture during inducer feed is about 10 g/L.
 14. The method of claim 1, comprising continuously adding the inducer to the culture for about 2 to about 8 hours after initiation of the method.
 15. The method of claim 14, comprising continuously adding the inducer to the culture for about 3 to about 6 hours after initiation.
 16. The method of claim 1, comprising isolating the recombinant protein about 2 to about 8 hours after starting to add the inducer to the culture.
 17. The method of claim 16, comprising isolating the recombinant protein about 3 to about 6 hours after starting to add the inducer to the culture.
 18. The method of claim 1, wherein the inducer is arabinose.
 19. The method of claim 1, comprising continuously adding the carbon source to the culture before induction.
 20. The method of claim 1, comprising continuously adding the carbon source to the culture before and during induction.
 21. The method of claim 1, comprising continuously adding the carbon source to the culture while the inducer is continuously added to the culture.
 22. The method of claim 1, comprising continuously adding the carbon source to the culture until the cell density of the culture achieves an OD₆₀₀ of about 70 to about
 110. 23. The method of claim 1, wherein the carbon source is a sugar-based carbon source.
 24. The method of claim 23, wherein the sugar-based carbon source is glucose.
 25. The method of claim 24, wherein the inducer is arabinose.
 26. The method of claim 25, comprising continuously adding the glucose to the culture medium while the arabinose is continuously added to the culture medium.
 27. The method of claim 25, comprising continuously adding the glucose until the cell density of the culture achieves an OD₆₀₀ of about
 80. 28. A method for producing a recombinant protein comprising: a) introducing into a bacterial host cell an expression vector encoding a recombinant protein under the control of an inducible promoter to form a recombinant bacterial cell; b) introducing the recombinant bacterial cell into a culture medium to form a cell culture; c) adding a carbon source to the cell culture as a continuous feed; d) monitoring cell growth in the cell culture for achievement of a threshold optical density (OD₆₀₀); e) adding an inducer of the inducible promoter to the cell culture as a continuous feed once the threshold optical density (OD₆₀₀) is achieved; and f) isolating the recombinant protein from the cell culture.
 29. The method of claim 28, comprising continuously adding the inducer to the culture when the cell density of the culture achieves an OD₆₀₀ of about 70 to about
 110. 30. The method of claim 29, comprising continuously adding the inducer to the culture when the cell density of the culture achieves an OD₆₀₀ of about 70 to about
 105. 31. The method of claim 30, comprising continuously adding the inducer to the culture when the cell density of the culture achieves an OD₆₀₀ of about 75 to about
 85. 32. The method of claim 31, comprising continuously adding the inducer to the culture when the cell density of the culture achieves an OD₆₀₀ of about
 80. 33. The method of claim 28, wherein the inducer is added to the culture at a constant rate, the inducer is added to the culture by DO-stat feed or the inducer is added to the culture by pH-stat feed.
 34. The method of claim 33, wherein the inducer is added to the culture at a constant rate of from about 3.35 g/L/h to about 16 g/L/h, the inducer is added to the culture by DO-stat feed or the inducer is added to the culture by pH-stat feed.
 35. The method of claim 28, wherein the total amount of inducer added to the culture is from about 4 g/L to about 40 g/L.
 36. The method of claim 35, wherein the total amount of inducer added to the culture is from about 5 g/L to about 20 g/L.
 37. The method of claim 36, wherein the total amount of inducer added to the culture is from about 7 g/L to about 15 g/L.
 38. The method of claim 37, wherein the total amount of inducer added to the culture is about 10 g/L.
 39. The method of claim 28, comprising isolating the recombinant protein from the culture about 2 hours to about 8 hours after initiation of inducer feed.
 40. The method of claim 39, comprising isolating the recombinant protein from the culture about 3 hours to about 6 hours after initiation of inducer feed.
 41. The method of claim 28, wherein the carbon source is added to the cell culture during constant rate feed of both the carbon source and the inducer, during DO-stat feed of both the carbon source and the inducer or during pH-stat feed of both the carbon source and the inducer.
 42. The method of claim 28, wherein the inducer is arabinose.
 43. The method of claim 28, wherein the carbon source is a sugar-based carbon source.
 44. The method of claim 43, wherein the sugar-based carbon source is glucose.
 45. The method of claim 28, wherein the carbon source is glucose and the inducer is arabinose.
 46. The method of claim 45, comprising continuously adding glucose to the cell culture while the arabinose is continuously added to the cell culture.
 47. The method of claim 45, comprising continuously adding glucose to the cell culture until the cell density of the cell culture achieves an OD₆₀₀ of about
 80. 48. A method for producing a recombinant protein comprising: culturing a recombinant bacterial cell to express a recombinant protein by continuously adding an inducer to a culture comprising the bacterial cell after the culture achieves a threshold parameter, wherein the bacterial cell comprises a nucleic acid sequence corresponding to a gene of N. meningitidis serogroup B.
 49. The method of claim 48, wherein the threshold parameter is optical density (OD), dissolved oxygen (DO), pH, concentration of nutrient in culture medium, total concentration of carbon source added to culture medium, or any combination thereof.
 50. The method of claim 49, wherein the threshold parameter is optical density (OD) of the culture.
 51. The method of claim 50, wherein the continuous inducer feed is initiated when the cell density of the culture achieves an OD₆₀₀ of about 70 to about
 110. 52. The method of claim 51, wherein the continuous inducer feed is initiated when the cell density of the culture achieves an OD₆₀₀ of about 70 to about
 105. 53. The method of claim 52, wherein the continuous inducer feed is initiated when the cell density of the culture achieves an OD₆₀₀ of about 75 to about
 85. 54. The method of claim 53, wherein the continuous inducer feed is initiated when the cell density of the culture achieves an OD₆₀₀ of about
 80. 55. The method of claim 48, wherein the inducer is added to the culture at a constant rate of about 3.35 g/L/h to about 16 g/L/h, the inducer is added by DO-stat feed or the inducer is added by pH-stat feed.
 56. The method of claim 48, wherein the total amount of inducer added to the culture during induction is from about 4 g/L to about 40 g/L.
 57. The method of claim 56, wherein the total amount of inducer added to the culture during induction is from about 5 g/L to about 20 g/L.
 58. The method of claim 57, wherein the total amount of inducer added to the culture medium during induction is about 7 g/L to about 15 g/L.
 59. The method of claim 58, wherein the total amount of inducer added to the culture during induction is about 10 g/L.
 60. The method of claim 48, wherein the inducer feed continues for about 2 hours to about 8 hours after initiation of the method.
 61. The method of claim 60, wherein the inducer feed continues for about 3 hours to about 6 hours after initiation of the method.
 62. The method of claim 48, wherein the recombinant protein is harvested from about 2 hours to about 8 hours after initiation of inducer feed.
 63. The method of claim 62, wherein the recombinant protein product is isolated from about 3 hours to about 6 hours after initiation of inducer feed.
 64. The method of claim 48, wherein the carbon source feed to the culture medium continues during inducer feed to the culture.
 65. The method of claim 48, wherein the inducer is arabinose.
 66. The method of claim 48, wherein the carbon source is a sugar-based carbon source.
 67. The method of claim 66, wherein the sugar-based carbon source is glucose.
 68. The method of claim 67, wherein the inducer is arabinose.
 69. The method of claim 68, wherein glucose is fed to the culture at a constant feed rate during the constant rate feed of arabinose to the culture, during DO-stat of both the glucose and the inducer or by pH-stat feed of both the glucose and the inducer.
 70. The method of claim 68, wherein glucose is fed to the culture at a constant feed rate during the constant rate feed of arabinose to the culture, by DO-stat feed of both the glucose and the inducer until the cell density in the culture achieves an OD₆₀₀ of about 80 or by pH-stat feed of both the glucose and the inducer until the cell density in the culture achieves an OD₆₀₀ of about
 80. 71. The method of claim 68, wherein the recombinant meningococcal 2086 protein comprises a lipoprotein (rLP2086).
 72. The method of claim 68, wherein the protein comprises a non-lipidated protein.
 73. The method of claim 68, wherein the protein comprises meningococcal 2086 subfamily A protein.
 74. The method of claim 68, wherein the protein comprises meningococcal 2086 subfamily B protein.
 75. A method for producing a recombinant meningococcal 2086 protein (P2086) comprising: (a) introducing into a bacterial host cell an expression vector encoding a recombinant 2086 protein under the control of an inducible promoter to form a recombinant bacterial cell; (b) introducing the recombinant bacterial cell to a culture medium to form a culture; (c) adding a carbon source to the culture; (d) monitoring cell growth in the culture for achievement of a threshold optical density (OD); (e) continuously adding an inducer of the inducible promoter to the culture once the cell density of the culture achieves an optical density of about 70 to 110; and (f) isolating the recombinant meningococcal 2086 protein from the culture after about 3 hours to about 6 hours after commencement of continuously adding the inducer.
 76. A composition comprising: a bacterial culture comprising a recombinant meningococcal 2086 protein at a density of at least about 1.5 g/L based on the total volume of the bacterial culture.
 77. The composition of claim 76, wherein the recombinant meningococcal 2086 protein is lipidated protein.
 78. The composition of claim 76, wherein the recombinant meningococcal 2086 protein is non-lipidated protein.
 79. The composition of claim 76, wherein the recombinant meningococcal 2086 protein is a 2086 subfamily A protein.
 80. The composition of claim 76, wherein the recombinant protein is a 2086 subfamily B protein.
 81. The composition of claim 80, wherein the culture comprises a second recombinant meningococcal 2086 protein.
 82. The composition of claim 81, wherein the second 2086 recombinant meningococcal protein is a 2086 subfamily A protein.
 83. The composition of claim 76, wherein the density of the recombinant meningococcal 2086 protein is at least about 1.7 g/L based on the total volume of the bacterial culture.
 84. The composition of claim 76, wherein the density of the recombinant meningococcal 2086 protein is at least about 2.0 g/L based on the total volume of the bacterial culture.
 85. The composition of claim 76, wherein the density of the recombinant meningococcal 2086 protein is at least about 3.0 g/L based on the total volume of the bacterial culture.
 86. A composition comprising: a bacterial culture comprising a recombinant meningococcal 2086 protein prepared according to the process comprising culturing a recombinant bacterial cell to express a recombinant protein comprising continuously adding a carbon source to a culture comprising the recombinant bacterial cell and continuously adding an inducer to the culture after the culture achieves a threshold parameter, and isolating the recombinant protein from the culture. 